The present invention relates to methods of preventing or reducing pathogenesis of oxidant stress-associated diseases of the aerodigestive tract. More particularly, the present invention relates to methods of employing hydroxocobalamin (vitamin B12a, OHxe2x80x94CO), deferoxamine (DES) and reduced glutathione (GSH) to reduce or prevent tobacco smoke (TS)-induced cellular or macromolecular damage in the aerodigestive tract.
Harmful Effects of Tobacco Consumption:
The deleterious effects of tobacco abuse, principally via cigarette smoking, are well known. Tobacco is a worldwide public health hazard accounting for significant morbidity and mortality. Although smoking places an abundant oxidant insult to the oropharynx and respiratory tract, the oxidant burden is deleterious to the entire body of the tobacco consumer. Namely, tobacco consumption leads to development or enhancement of atherosclerosis, cardiovascular disease, chronic obstructive pulmonary disease and various forms of cancer, including carcinomas of the mouth, pharynx, esophagus and lung.
There are three principal ways to consume tobacco: smoking, chewing and dipping and snuffing. Approximately 50 million Americans smoke and countless others are affected by tobacco smoke (TS) as secondary smokers. Children of smokers breathe this second-hand smoke and have more respiratory problems than children of non-smokers. Smokeless tobacco is used by as many as 12 million individuals and has a detrimental effect on the oral cavity plus systemic effects from buccal absorption of nicotine and other chemicals. Chewing looseleaf tobacco and xe2x80x9cdippingxe2x80x9d moist, ground snuff tobacco are common uses of tobacco without smoking.
Studies have estimated that TS has over 3,000 different constituents, of which many are toxic, carcinogenic and/or generate free radical species. Free radicals are atoms or molecules containing an unpaired electron. Oxygen free radicals include the superoxide free radical (.O2xe2x88x92) and the hydroxyl radical (OH.) which, together with hydrogen peroxide (H2O2) and singlet oxygen (1O2), are jointly called reactive oxygen species (ROS). Due to their high reactivity they may lead to chemical modification and impairment of the components of living cells, such as proteins, lipids, carbohydrates and nucleotides.
Tobacco smoke mediated oxidant injury is similar to that induced by smog, thereby increasing such noxious stimuli to primary and secondary smokers in polluted atmospheric environments.
Most of constituents of TS have been identified in so-called mainstream and side stream TS. The former is that volume of smoke drawn through the mouthpiece of the tobacco product during puffing while side stream smoke is that smoke emitted from the smoldering cigarette in between puffs. Although tar and nicotine are retained in the filter of cigarettes, this applies mainly to mainstream smoke, when comparing filter and non-filter cigarettes. Mainstream smoke emission is also markedly reduced both in low and in ultra low tar yield cigarettes. However, the emissions of toxic and carcinogenic components in side stream smoke are not significantly reduced in filter cigarettes when compared to non-filter counterparts. Thus, side stream smoke is a major contributor to environmental smoke, affecting both the smoker and their non-smoking counterparts, so called secondary smokers.
Evidence shows that cigars as well as cigarettes are highly toxic and addictive. Tobacco smokers have a similar increased risk for oral and laryngeal cancers. Evidence indicates that one cigar generates levels of carcinogenic particles exceeding those generated by three cigarettes. Fumes from cigars are also of greater consequence to secondary smokers. Epidemiologic studies reveal greater frequencies of heart disease, emphysema, and cancers of the mouth and pharynx in cigar smokers when compared to matched non-smokers. Cigar smokers may spend one full hour smoking a single large cigar and commonly hold an unlit cigar in the mouth, allowing further exposure to toxins by local absorption. Thus, consumption of cigars may produce an equal or greater burden of toxic exposure to TS than cigarettes. Recently, sales of cigars have risen, partly due to their gaining popularity with women and the advent of the female friendly xe2x80x9ccigar barxe2x80x9d.
Oral Diseases Associated With Tobacco Consumption: Tobacco, whether smoked or chewed, causes common untoward effects in the oral cavity. Tobacco smoke has two chances to exert its deleterious effects in the mouth; when it is inhaled by the smoker and on its exit during exhalation.
Over 30,000 new cases of cancer of the oral cavity are diagnosed annually, accounting for 2-4 percent of all new cancers. Oral cancer kills 8,000 patients each year and only half of cases diagnosed annually have a five-year survival. The great majority of these patients are users of tobacco products. Oral squamous cell carcinoma (SCC) is the most common malignancy of the head and neck with a worldwide incidence of over 300,000 new cases annually. The disease is characterized by a high rate of morbidity and mortality (approximately 50%) and in this respect is similar to malignant melanoma (1-4). The major inducer of oral SCC is exposure to tobacco which is considered to be responsible for 50-90% of cases world-wide (5, 6). As such, the incidence of oral SCC in tobacco smokers is 4-7 times higher than in non-smokers (7, 8). Moreover, the higher TS-related risk for oral SCC is manifested by a reduction in the mean age of development of the disease by 15 years as compared to non-smokers (9).
Leukoplakia, a tobacco induced white patch on the buccal mucosa, as found in smokers, is a localized irritation due to direct contact of smoked or smokeless tobacco and it is directly related to the frequency and years of tobacco abuse. Although leukoplakia is a benign oral lesion, it has a malignant potential.
In addition, tobacco contributes to other oral symptoms or pathologies of the mouth and teeth. Tobacco may cause halitosis, may numb the taste buds, and interfere with the smell and the taste of food. It may stain teeth and contribute to dental caries. Smokers have more dental tartar (calculus) than non-smokers. Tobacco is associated also with destructive periodontal (gum) disease and tooth loss. Acute necrotizing ulcerative gingivitis (xe2x80x9ctrench mouthxe2x80x9d) is a destructive, painful inflammatory condition occurring mainly in tobacco smokers. Swelling of the nasal and sinus membranes have also been associated, purportedly, in individuals who are xe2x80x9callergicxe2x80x9d to TS.
Oral submucous fibrosis occurs mainly in India and is a chronic, progressive premalignant condition. The etiology is chronic chewing of tobacco or areca nut or both. The fibrosis results in restriction of mouth opening and involves the palates, tonsillar fossa, buccal mucosa and underlying muscle. Associated with this condition is also oropharyngeal carcinomas, also with a high frequency in India and associated in 70% of cases with chewing tobacco. Smokeless tobacco and areca nut usage is also common in Pakistan, Bangladesh and Java and in these and Indian immigrants to the United States and United Kingdom.
Tobacco smoke also affects the skin adversely. Dr. Douglas Model of England in 1985 added to the medical lexicon the term xe2x80x9csmoker""s facexe2x80x9d from a study with pictures of 116 cases and suitable non-smoking controls (10). Akin to photodamage, those with smoker""s face appear older and have more wrinkles.
Molecular Damage Resulting From Exposure to TS: Tobacco smoke induces oxidative damage to lipids, DNA and proteins, particularly via protein-SH groups as a consequence of containing high levels of both free radicals as well as aldehydes, including acetaldehyde (ethanol), propanol and acrolein, as well as other deleterious molecules.
Oxidant Injury: Tobacco smoke is divided into two phases; tar and gas-phase smoke. Tar contains high concentrations of free radicals. Many tar extracts and oxidants are water-soluble and reduce oxygen to superoxide radical which can dismutate to form the potent oxidant H2O2. Oxidants in gas-phase smoke are reactive carbon- and oxygen-centered radicals with extremely short half lives.
Cells subjected to oxidative stress develop severely affected cellular function and suffer damage to membrane lipids, to proteins, to cytoskeletal structures and to DNA. Free radical damage to DNA has been measured as formation of single-strand breaks, double-strand breaks and chromosomal aberrations. Cells exposed to ionizing radiation and TS have also been demonstrated to have an increased intracellular DNA damage, a precursor of mutations and development of malignancies. It has been shown that TS elicits protein carbonylation in plasma and that, in contrast, exposure of human plasma to gas-phase but not to whole TS produces oxidative damage to lipids.
Redox-Active Metals: Redox-active metal ions, such as iron and copper, in the presence of H2O2 and other low-reactive free radicals found in TS, such as superoxide radicals, participate in the deleterious Haber-Weiss and Fenton reactions, in which the highly reactive hydroxyl free radicals are produced.
Aldehydes: Studies have shown that exposure of plasma to TS results in protein damage in the form of protein carbonylation (11) and in oxidation of plasma lipids and antioxidants (12). The source of the accumulation of protein carbonylation was found to be due to aldehydes present in TS (13, 14). In addition, it was shown that several salivary enzymes such as amylase, lactic dehydrogenase (LDH), and acid phosphatase were considerably affected by TS (14, 15), where both TS-based aldehydes, such as acrolein and crotonaldehyde, as well as oxygen free radicals were implicated as the causative agents affecting the above enzymes (14, 15).
Physiological Antioxidants:
Glutathione: Glutathione, a sulfur-containing tripeptide (L-xcex3-glutamyl-1-cysteine-glycine) is the most abundant non-protein thiol in mammalian cells and is recognized as the primordial antioxidant. Glutathione, in its reduced form, xe2x80x9cGSHxe2x80x9d, acts as a substrate for glutathione-S-transferase and glutathione peroxidase, enzymes catalyzing reactions involved in detoxification of xenobiotic compounds and in antioxidation of ROS and other free radicals. This ubiquitous protein plays a vital function in maintaining the integrity of free radical sensitive cellular components. Under states of GSH depletion, including malnutrition and severe oxidative stress, cells may then become injured from excess free radical damage and die.
Oral Peroxidase: Oral peroxidation is the pivotal enzymatic activity of the salivary antioxidant system (16-19). Oral peroxidase activity is composed of the combined activity of two peroxidases, salivary peroxidase (SPO) and myeloperoxidase (MPO). Salivary peroxidase, which is secreted by the major salivary glands, mainly the parotid gland (18), contributes 80% of the total oral peroxidase (OPO) activity, while MPO, produced by leukocytes (20), contributes the remaining 20% of OPO activity. Oral peroxidase performs two functions preventing oxidant injury; it reduces the level of H2O2 excreted into the oral cavity from the salivary glands, by bacteria and by leukocytes, and it inhibits the metabolism and proliferation of various bacteria in the oral cavity.
Oral peroxidase is involved in destroying TS-associated H2O2. Tobacco smoke-associated hydrocyanic acid (HCN) is metabolized by the liver to thiocyanate ion (SCNxe2x88x92). This SCNxe2x88x92 is specifically sequestered from the plasma by the parotid gland and is secreted by this gland into the oral cavity. Its concentration in the saliva of non-smokers ranges from 0.3-1.5 mM, while the respective range in smokers is approximately 1.4-4.0 mM, depending on the number of cigarettes smoked per day, with a prolonged t1/2 of 9.5 h (21). Following its secretion in saliva, SCNxe2x88x92 reacts with, and eliminates H2O2 in the oral cavity in a reaction catalyzed by OPO, as described in FIG. 2a. However, it has been shown that if OPO is damaged or depleted, as occurs upon exposure to TS, the H2O2 in the oral cavity is not eliminated and remains available for further reaction with redox-active metal ions which are secreted via the parotid gland saliva (22, 23).
In the reaction of SCNxe2x88x92+H2O2xe2x86x92OSCNxe2x88x92+H2O, which is catalyzed by OPO, H2O2 oxidizes SCNxe2x88x92, a detoxification product of cyanide secreted mainly by the parotid gland. In this reaction, SCNxe2x88x92 acts as the electron-donating component, similarly to GSH in other biological systems (20, 24, 25). Two potent antibacterial oxidizing products evolve from this reaction: hydrogen hypothiocyanite (HOSCN) and its conjugated anion, OSCNxe2x88x92. The antibacterial activity of HOSCN and OSCNxe2x88x92 stems from their ability to react with sulfhydryl groups of bacterial enzymes that are vital for glycolysis, such as hexokinase, aldolase and pyruvate kinase (20, 25-28).
The importance of OPO in oral disease prevention has been demonstrated in several studies. For example, studies using animal models or the Ames test have shown that saliva inhibits the mutagenicity of known oral cancer inducers, such as TS, 4NQO and benzopyrene (29, 30). Biochemical studies have also demonstrated that saliva inhibits production of ROS such as superoxide free radical and H2O2 from betel quid tobacco, the most potent inducer of oral cancer (31). These observations are further supported in the observation that patients with oral lichen planus, a premalignant lesion, have reduced salivary antioxidant capacity (32).
Several prior art approaches have been employed in order to reduce or prevent incidence of oral disease resulting from oxidant injury.
For example, cigarette filters are used to trap TS tar but do not the gas-phase compounds.
One approach has employed a filter for TS providing chemo-sorptive properties to reduce aldehyde concentration in TS (33).
Another approach has employed oral megadoses of antioxidants in attempts to reduce generation of H2O2 resulting from the xe2x80x9crespiratory burstxe2x80x9d reaction associated with phagocytic activity of macrophages and neutrophils. It has been shown that smokers have a higher xe2x80x9crespiratory burstxe2x80x9d reaction than non-smokers and that this may be associated with the increased incidence of aerodigestive tract disease in the former.
In yet another approach, dipeptide compounds with pharmaceutical properties to increase glutathione levels were employed (34).
A further approach utilized a glycine carboxylic acid alkyl mono-ester of glutathione to increase cellular GSH levels (35).
In yet a further approach, administration of a combination of glutathione and selenium was suggested for preventing oxidant injury resulting from exposure to TS (36).
In another approach, administration of a combination of glutathione, ascorbic acid, selenium and a sulfur-containing amino acid was suggested in order to prevent oral oxidant injury (37).
In yet another approach, administration of a combination including some or all of the following antioxidants; L-glutathione, L-selenomethionine, L-selenocysteine, ascorbyl palmitate, ascorbic acid esters, L-cysteine, N-acetyl-l-cysteine, tocopherol acetate, tocopherol succinate, vitamin A, a zinc salt, methionine and taurine was suggested in order to provide intra-oral protection from oxidant injury (38).
All of the aforementioned approaches, however, failed to demonstrate prevention or reduction of loss of OPO activity or HCNxe2x88x92-, redox-active metal ion- or aldehyde-induced cell death or protein carbonylation resulting from TS-associated oxidative stress, processes being further described in the Examples section below.
There is thus a widely recognized need for, and it would be highly advantageous to have, methods of preventing or reducing TS-associated oxidant injury in the aerodigestive tract devoid of the above limitation.
According to the present invention there is provided a pharmaceutical composition for preventing or reducing tobacco smoke-associated injury in the aerodigestive tract of a subject, the pharmaceutical composition comprises, as an active ingredient, a therapeutically effective amount of an agent capable of preventing or reducing tobacco smoke-associated decrease in peroxidase activity in the aerodigestive tract of the subject, and a pharmaceutically acceptable carrier.
According to further features in preferred embodiments of the invention described below, the agent comprises a cyanide chelator.
According to still further features in the described preferred embodiments, the agent is hydroxocobalamin.
According to still further features in the described preferred embodiments, the pharmaceutically acceptable carrier is selected so as to enable administration of the pharmaceutical composition by a route selected from the group consisting of the intranasal, transdermal, intradermal, oral, buccal, parenteral, topical, rectal and inhalation route.
According to still further features in the described preferred embodiments, the the pharmaceutical composition is formulated as a solution, suspension, emulsion or gel.
According to still further features in the described preferred embodiments, the pharmaceutical composition further includes a formulating agent selected from the group consisting of a suspending agent, a stabilizing agent and a dispersing agent.
According to another aspect of the present invention there is provided a pharmaceutical composition for preventing or reducing tobacco smoke-associated injury in the digestive tract of a subject, the pharmaceutical composition comprises, as an active ingredient, a therapeutically effective amount of an agent capable of preventing or reducing tobacco smoke-associated death of cells in the digestive tract of the subject, and a pharmaceutically acceptable carrier.
According to further features in preferred embodiments of the invention described below, the cells are lymphocytes.
According to still further features in the described preferred embodiments, the agent comprises an iron chelator.
According to still further features in the described preferred embodiments, the agent is deferoxamine.
According to still further features in the described preferred embodiments, the pharmaceutically acceptable carrier is selected so as to enable administration of the pharmaceutical composition by a route selected from the group consisting of the intranasal, transdermal, intradermal, oral, buccal, parenteral, topical, rectal and inhalation route.
According to still further features in the described preferred embodiments, the the pharmaceutical composition is formulated as a solution, suspension, emulsion or gel.
According to still further features in the described preferred embodiments, the pharmaceutical composition further includes a formulating agent selected from the group consisting of a suspending agent, a stabilizing agent and a dispersing agent.
According to yet another aspect of the present invention there is provided a pharmaceutical composition for reducing or preventing tobacco smoke-associated injury in the aerodigestive tract of a subject, the pharmaceutical composition comprises, as an active ingredient, a therapeutically effective amount of an agent capable of preventing or reducing tobacco smoke-associated death of cells in the aerodigestive tract of the subject, and a pharmaceutically acceptable carrier.
According to further features in preferred embodiments of the invention described below, the cells are lymphocytes.
According to still further features in the described preferred embodiments, the agent comprises an antioxidant.
According to still further features in the described preferred embodiments, the agent is glutathione.
According to still another aspect of the present invention there is provided an oral composition in the form of a toothpaste, powder, liquid dentifrice, mouthwash, denture cleanser, chewing gum, lozenge, paste, gel or candy comprising, as an active ingredient, an agent capable of reducing or preventing tobacco smoke-associated loss of peroxidase activity in the aerodigestive tract of a subject.
According to still further features in the described preferred embodiments, the oral composition further comprising at least one flavorant selected from the group consisting of wintergreen oil, oregano oil, bay leaf oil, peppermint oil, spearmint oil, clove oil, sage oil, sassafras oil, lemon oil, orange oil, anise oil, benzaldehyde, bitter almond oil, camphor, cedar leaf oil, marjoram oil, citronella oil, lavendar oil, mustard oil, pine oil, pine needle oil, rosemary oil, thyme oil, and cinnamon leaf oil.
According to an additional aspect of the present invention there is provided an oral composition in the form of a toothpaste, powder, liquid dentifrice, mouthwash, denture cleanser, chewing gum, lozenge, paste, gel or candy comprising, as an active ingredient, an agent capable of reducing or preventing tobacco smoke-associated death of cells in the digestive tract of a subject.
According to yet an additional aspect of the present invention there is provided an oral composition in the form of a toothpaste, powder, liquid dentifrice, mouthwash, denture cleanser, chewing gum, lozenge, paste, gel or candy comprising, as an active ingredient, an agent capable of reducing or preventing tobacco smoke-associated death of cells in the aerodigestive tract of a subject.
According to still an additional aspect of the present invention there is provided a filter comprising an agent being capable of reducing or preventing tobacco smoke-associated loss of peroxidase activity in the aerodigestive tract of a subject, the filter being designed and configured so as to enable release of said agent therefrom when in use by the subject.
According to further features in preferred embodiments of the invention described below, the filter being designed and configured as a tobacco smoke filter.
According to a further aspect of the present invention there is provided a filter comprising an agent being capable of reducing or preventing tobacco smoke-associated death of cells in the digestive tract of a subject, the filter being designed and configured so as to enable release of said agent therefrom when in use by the subject.
According to yet a further aspect of the present invention there is provided a filter comprising an agent being capable of reducing or preventing tobacco smoke-associated death of cells in the aerodigestive tract of a subject, the filter being designed and configured so as to enable release of said agent therefrom when in use by the subject.
According to still a further aspect of the present invention there is provided a filter comprising an agent being capable of reducing or preventing tobacco smoke-associated loss of peroxidase activity in the aerodigestive tract of a subject, the filter being designed and configured so as to enable physico-chemical interaction between said agent and said tobacco smoke when in use by the subject.
According to the present invention there is provided a filter comprising an agent being capable of reducing or preventing tobacco smoke-associated death of cells in the digestive tract of a subject, the filter being designed and configured so as to enable physico-chemical interaction between said agent and said tobacco smoke when in use by the subject.
According to another aspect of the present invention there is provided a filter comprising an agent being capable of reducing or preventing tobacco smoke-associated death of cells in the aerodigestive tract of a subject, the filter being designed and configured so as to enable physico-chemical interaction between said agent and said tobacco smoke when in use by the subject.
According to yet another aspect of the present invention there is provided method of reducing or preventing a tobacco smoke-associated decrease in peroxidase activity in the aerodigestive tract of a subject, the method comprising administering to the subject hydroxocobalamin, in an amount effective for preventing or reducing the tobacco smoke-associated decrease in peroxidase activity in the aerodigestive tract of the subject.
According to still another aspect of the present invention there is provided a method of reducing or preventing a tobacco smoke-associated death of cells in the digestive tract of a subject, the method comprising administering to the subject deferoxamine, in an amount effective for preventing or reducing the tobacco smoke-associated death of cells in the digestive tract of the subject.
According to further features in preferred embodiments of the invention described below, the administering is effected locally or systemically.
According to still further features in the described preferred embodiments, the administering is effected into the oral cavity.
According to an additional aspect of the present invention there is provided a method of reducing or preventing a tobacco smoke-associated death of cells in the aerodigestive tract of a subject, the method comprising administering to the subject glutathione, in an amount effective for preventing or reducing the tobacco smoke-associated death of cells in the aerodigestive tract of the subject.
According to yet an additional aspect of the present invention there is provided a smoking product comprising an agent being capable of reducing or preventing tobacco smoke-associated loss of peroxidase activity in the aerodigestive tract of a subject, the smoking product being designed and configured so as to enable release of said agent therefrom when in use by the subject.
According to still an additional aspect of the present invention there is provided a smoking product comprising an agent being capable of reducing or preventing tobacco smoke-associated death of cells in the digestive tract of a subject, the smoking product being designed and configured so as to enable release of said agent therefrom when in use by the subject.
According to a further aspect of the present invention there is provided a smoking product comprising an agent being capable of reducing or preventing tobacco smoke-associated death of cells in the aerodigestive tract of a subject, the smoking product being designed and configured so as to enable release of said agent therefrom when in use by the subject.
According to yet a further aspect of the present invention there is provided a smoking product comprising an agent being capable of reducing or preventing tobacco smoke-associated loss of peroxidase activity in the aerodigestive tract of a subject, the smoking product being designed and configured so as to enable physico-chemical interaction between said agent and said tobacco smoke when in use by the subject.
According to still a further aspect of the present invention there is provided a smoking product comprising an agent being capable of reducing or preventing tobacco smoke-associated death of cells in the digestive tract of a subject, the smoking product being designed and configured so as to enable physico-chemical interaction between said agent and said tobacco smoke when in use by the subject.
According to the present invention there is provided a smoking product comprising an agent being capable of reducing or preventing tobacco smoke-associated death of cells in the aerodigestive tract of a subject, the smoking product being designed and configured so as to enable physico-chemical interaction between said agent and said tobacco smoke when in use by the subject.
The present invention successfully addresses the shortcomings of the presently known configurations by providing methods of preventing or reducing tobacco smoke-associated injury in the aerodigestive tract.